We have used digital imaging microscopy to evaluate possible guidance mechanisms of mouse neuroblastoma cells cultured on microfabricated glass substrates. Substrates were bound with either small charged amine or uncharged alkane molecules using silane-coupling chemistry or covered with proteins (laminin, collagen, fibronectin, albumin) by adsorption. Photolithographic procedures were used to pattern substrates with amine - alkane, amine - protein and alkane - protein combinations. Interference reflection microscopy (IRM) was used to visualize sites of cellular attachment on substrates (focal and close contacts), and image processing techniques were used to quantify the results in three different ways: total cellular area in contact with a substrate, area corresponding to focal and close contacts, and the percentage of focal and close contacts on each substrate. Cells attached to and extended neurites on each of the substrates we tested. IRM images of growth cones displayed similar gray levels on amine, alkane, fibronectin and albumin substrates, whereas images on laminin and collagen were brighter. Brightness on laminin substrates was correlated with less area of focal contact and greater area with no contact. When cells were provided with choices on patterned substrates, they displayed the following preference of attachment: laminin, fibronectin, collagen > amine > alkane > albumin. This hierarchy reflected greater total surface area on preferred substrates. There was, however, no correlation between the hierarchy and the area corresponding to focal and close contacts or the percentage of contacts on different substrates. Our results support several recent observations which demonstrated that guidance was not correlated with differential adhesivity. These results are more in line with the theory that guidance is controlled by a signal transduction mechanism that couples locomotion with activation of membrane receptors.
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